CN111073983A - SNP marker related to identification of northern subspecies and Florida subspecies of largemouth bass and application thereof - Google Patents
SNP marker related to identification of northern subspecies and Florida subspecies of largemouth bass and application thereof Download PDFInfo
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Abstract
The invention discloses a SNP marker related to identification of northern sub species and Florida sub species of micropterus salmoides and application thereof, wherein the SNP marker consists of a 202 nd SNP locus of a genome sequence SEQ ID NO:1 of a micropterus salmoides NCCRP-1 gene and insertion deletion mutation loci of 76 th and 132 th and 133 th basic groups, and the three loci are completely linked. The SNP locus has the advantages of strong purposiveness and high accuracy, is simple to operate, is rapid to detect, is convenient to widely popularize and use, rapidly identifies northern subspecies of micropterus salmoides and Florida subspecies, and provides reliable basis for stock preservation and germplasm improvement.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to an SNP marker related to identification of northern subspecies of micropterus salmoides and Florida subspecies and application thereof.
Background
Lateolabrax japonicus (Micropterus Salmoides L.), commonly known as Lateolabrax japonicus, originally produced in fresh water lakes and rivers in North America, has the advantages of no muscle spurs, delicious meat, rapid growth, short culture period and the like, and is one of important freshwater cultured fishes. At its origin, Micropterus Salmoides are composed primarily of two subspecies, including Micropterus Floridanus subspecies distributed in the peninsula florida of the united states and northern Micropterus Salmoides distributed throughout the central and eastern regions of the united states, the northeast region of mexico, and the southeast region of canada. At the end of the 70 s of the 20 th century, major black bass is introduced for the first time in China, and with the development of time, most areas in China are cultivated. In 2010, the Zhujiang aquatic research institute of the Chinese aquatic science institute introduced northern subspecies of Lateolabrax japonicus and the Florida subspecies from the United states again, and both the northern subspecies and the Florida subspecies have good production performance. However, the external forms of the two subspecies are very similar, so that the two subspecies are difficult to distinguish from each other in appearance, and in addition, the two subspecies have natural hybridization propagation to generate hybrid offspring, so that the distinguishing and distinguishing difficulty of the micropterus salmoides germplasm is increased. How to accurately identify northern subspecies and Florida subspecies of the micropterus salmoides is a key for developing micropterus salmoides germplasm resource research and guaranteeing cultivation production.
In the prior art, the classification status and the germplasm source of different population of micropterus salmoides cannot be distinguished and identified effectively and accurately from the external morphology, so that the identification of two subspecies of micropterus salmoides is carried out by means of molecular biology at present. Early researches on identification of micropterus salmoides are mostly focused on RFLP, RAPD, isoenzyme specific sites and microsatellite molecular markers, the classification status of micropterus salmoides cultured in China is identified by the microsatellite molecular markers and the like, and the result shows that the micropterus salmoides cultured in China are considered to be northern subspecies.
In recent years, SNP molecular markers increasingly become the first choice genetic markers in genetic breeding research, and by combining the SNP molecular markers with identification of northern subspecies of micropterus salmoides, the northern subspecies of micropterus salmoides and the northern subspecies of Florida can be quickly and accurately identified, so that a related reliable basis is provided for reservation of the stock species of micropterus salmoides, interspecific hybridization and germplasm purification and rejuvenation.
Disclosure of Invention
According to the existing problems, the invention aims to provide the SNP marker related to identification of northern subspecies of micropterus salmoides and Florida subspecies and the application thereof.
The technical scheme adopted by the invention is as follows:
in a first aspect of the invention, a micropterus salmoides NCCRP-1 genome sequence such as SE1ID NO:1 is provided,
the 76 th mutation is A or base deletion,
the 132-th 133-bit mutation is AA or base deletion,
k at position 202 is a base G or T.
In a second aspect of the present invention, there is provided a method for identifying a SNP site or an indel mutation site related to a Perolabrax micropterus species, wherein the SNP site is located at the 202 nd position shown in the genomic sequence SEQ ID NO. 1 of the NCCRP-1 gene, and the indel mutation site is located at the 76 nd position and the 132 nd position 133 th position shown in the genomic sequence SEQ ID NO. 1.
In the third aspect of the invention, the application of the SNP site or the insertion deletion mutation site in identifying the largemouth bass species is provided.
According to an embodiment of the present invention, the micropterus salmoides are micropterus salmoides northern subspecies and florida subspecies.
In a fourth aspect of the present invention, there is provided a method for identifying northern subspecies of micropterus salmoides and northern subspecies of florida, comprising the steps of:
detecting that the SNP locus at the 76 th base of the genome sequence SEQ ID NO of the NCCRP-1 gene of the largemouth bass is AA or-genotype, wherein '-' represents the base deletion, if the base is '-', the fingered bass is the Florida subspecies, and if the base is AA, the northern subspecies;
or/and detecting that the SNP locus at the 132-133 th basic group of the genome sequence SEQ ID NO of the largemouth bass NCCRP-1 gene is AAAA or- - -genotype, wherein "- - -" represents basic group deletion, if the SNP locus is "AAAA", the SNP locus is Florida subspecies, and if the SNP locus is "- - - -", the SNP locus is northern subspecies;
or/and detecting that the SNP locus at the 202 nd base of the genome sequence SEQ ID NO:1 of the largemouth bass NCCRP-1 gene is GG or TT genotype, if GG, Florida subspecies, and if TT, North subspecies.
According to an embodiment of the present invention, the above method comprises the steps of:
1) extracting DNA of micropterus salmoides;
2) the extracted DNA is taken as a template, PCR is carried out to detect whether the genotypes of the 76 th site, the 132 th site and the 202 th site of the genome sequence SEQ ID NO:1 of the NCCRP-1 gene of the largemouth bass are AA or- -, AAAA or- - -, GG or TT genotypes respectively, wherein the "-" represents the base deletion.
According to the embodiment of the present invention, in step 2), the specific operation of PCR detection is:
carrying out PCR amplification on the DNA of the largemouth bass by using primers P1 and P2 to obtain a PCR product,
P1:5'-AAGCACAAAGTGTAGGTATTTTCCA-3'(SEQ ID NO:2)
P2:5'-GGCTGCGGATAAAAGCACTG-3'(SEQ ID NO:3)
and then sequencing and analyzing the obtained PCR product to determine whether the SNP locus genotype at the 202 nd site of the genome sequence of the largemouth bass NCCRP-1 gene is GG or TT genotype and whether the 76 nd and 132 nd and 133 nd sites of the insertion deletion mutation are AA or- - -, AAAA or- - -genotype respectively.
According to the embodiment of the invention, the PCR amplification reaction system is as follows:
according to the embodiment of the invention, the PCR amplification reaction program is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extension at 72 ℃ for 10 min.
The invention has the beneficial effects that:
(1) the method greatly reduces the artificial statistical difference when the conventional morphology identifies the micropterus salmoides, and can quickly identify the northern subspecies and the Florida subspecies of the micropterus salmoides.
(2) The identification method of northern subspecies of micropterus salmoides and Florida subspecies ensures the reliability of the detection result, does not need complicated operation, improves the treatment efficiency and the accuracy, and establishes a method for quickly and accurately identifying the northern subspecies of micropterus salmoides and the Florida subspecies. Compared with the traditional method, the method has the advantages of strong purposiveness, high accuracy, simple operation, quick detection and convenience for wide popularization and use.
Detailed Description
The genome sequence of the largemouth bass NCCRP-1 is shown as SEQ ID NO:1,
the 76 th mutation is A or base deletion,
the 132-th 133-bit mutation is AA or base deletion,
k at position 202 is a base G or T.
The largemouth bass species identifies related SNP sites or insertion deletion mutation sites, the SNP sites are positioned at the 202 nd site shown by the genomic sequence SEQ ID NO. 1 of the NCCRP-1 gene, and the insertion deletion mutation sites are positioned at the 76 th site and the 132 th site 133 shown by the SEQ ID NO. 1.
The SNP locus or the insertion deletion mutation locus is applied to identification of northern subspecies of micropterus salmoides and Florida subspecies.
A method for identifying northern subspecies and Florida subspecies of micropterus salmoides comprises the following steps:
detecting that the SNP locus at the 76 th base of the genome sequence SEQ ID NO of the NCCRP-1 gene of the largemouth bass is AA or-genotype, wherein '-' represents the base deletion, if the base is '-', the fingered bass is the Florida subspecies, and if the base is AA, the northern subspecies;
or/and detecting that the SNP locus at the 132-133 th basic group of the genome sequence SEQ ID NO of the largemouth bass NCCRP-1 gene is AAAA or- - -genotype, wherein "- - -" represents basic group deletion, if the SNP locus is "AAAA", the SNP locus is Florida subspecies, and if the SNP locus is "- - - -", the SNP locus is northern subspecies;
or/and detecting that the SNP locus at the 202 nd base of the genome sequence SEQ ID NO:1 of the largemouth bass NCCRP-1 gene is GG or TT genotype, if GG, Florida subspecies, and if TT, North subspecies.
Preferably, the above method comprises the steps of:
1) extracting DNA of micropterus salmoides;
2) using the extracted DNA as a template, PCR is carried out to detect whether the genotypes of the 76 th, 132 th and 202 nd sites of the genome sequence SEQ ID NO:1 of the NCCRP-1 gene of the largemouth bass are A-, AA-, GT-genotypes or not. Wherein "-" represents a base deletion.
Preferably, in the step 2), when detecting whether the genotypes of the genomic sequence SEQ ID NO. 1 at the 76 th site, the 132 th site and the 202 th site of the NCCRP-1 gene of the largemouth bass are AA or- -, AAAA or- - -, GG or TT genotypes, the PCR detection is performed by the following specific steps:
carrying out primary PCR amplification on the DNA of the largemouth bass by using primers P1 and P2 to obtain a primary PCR product,
P1:5'-AAGCACAAAGTGTAGGTATTTTCCA-3'(SEQ ID NO:2)；
P2:5'-GGCTGCGGATAAAAGCACTG-3'(SEQ ID NO:3)；
and then sequencing and analyzing the obtained PCR product to determine whether the genotypes of the 76 th, 132 th and 202 nd positions of the genome sequence of the NCCRP-1 gene of the largemouth bass are AA or- -, AAAA or- -, GG or TT genotypes respectively.
Preferably, the PCR amplification reaction system is:
the PCR amplification reaction program is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extension at 72 ℃ for 10 min.
The present invention will be further described with reference to the following specific examples.
Example 1 acquisition of SNP markers associated with identification of northern subspecies of Perciformis and Florida subspecies
The invention discovers that the genome sequence of the NCCRP-1 gene of the largemouth bass is shown as SEQ ID NO:1, wherein the 76 th mutation is A or base deletion, two genotypes AA and- - ("- -" represents base deletion) exist, the 132 th 133 th mutation is AA or base deletion, two genotypes AAAA and- - ("- - - -" represents base deletion) exist, the 202 nd K is base G or T, and two genotypes GG and TT exist.
In the experiment, 128 samples of micropterus salmoides from 5 groups (the subspecies classification status is determined) are adopted for subspecies identification. Population 1 is northern subintiage (21) of micropterus salmoides and 12 of micropterus sallowda (source: Zhujiang aquatic research institute), population 2 is northern subintiage (30) of micropterus sallowda reserved in Foshan Sanshui platinum base, population 3 is northern subintiage (10) of micropterus sallowda collected in 2017 and northern subintiage (11) of micropterus sallowda, population 4 is northern subintiage (20) of micropterus sallowda propagated in 2019 in Foshan Sanshui platinum base, population 5 is a northern micropterus sample collected in 2018, wherein the northern terus sallowda (12) and the northern perch florida (12) are provided.
(1) Extraction of genomic DNA
Shearing part of tail fin of Lateolabrax japonicus by 0.5 × 0.5cm with scissors sterilized with alcohol, and storing in anhydrous alcohol at room temperature. The fin ray genome DNA is extracted by a marine animal tissue genome DNA extraction kit (Tiangen). The quality of the genomic DNA was checked by electrophoresis on a 1.0% agarose gel and the concentration was determined by UV spectrophotometer (Eppendorf, AG2231 type) and stored at-20 ℃ for future use.
(2) The PCR amplification reaction system is as follows:
wherein P1 primer: 5'-AAGCACAAAGTGTAGGTATTTTCCA-3' (SEQ ID NO: 2);
p2 primer: 5'-GGCTGCGGATAAAAGCACTG-3' (SEQ ID NO: 3).
The PCR amplification reaction program is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extension at 72 ℃ for 10 min.
(3) Results
And (3) detecting the PCR product by 1.0% agarose gel electrophoresis, sending the PCR product to a company Limited in Biotechnology engineering (Shanghai) for sequencing, and counting the SNP sites and the peak patterns of the sequencing results. The SNP site of 202 nd base of the genomic sequence SEQ ID NO of largemouth bass NCCRP-1 is shown in Table 1.
TABLE 1 cases of different genotypes of 202 nd SNP of largemouth bass NCCRP-1 gene in 5 groups
Example 2 verification of the accuracy of SNP sites
The accuracy of the SNP site of example 1 was identified by the SNaPshot method, comprising the steps of:
primer sequence
According to a partial genome sequence of the NCCRP-1 gene of the largemouth bass, a SNaPshot extension primer is designed:
P3：5'-AACCAAATTTAAAACCAACCCCAAAAA-3'(SEQ ID NO.4)；
P4：5'-ctgactgactGTAATTATAGGTTTGATTTGAATGGACT-3'(SEQ ID NO.5)；
P5：5'-ctgactgactgactgactgAATGTGTTAAACGGGTTATTTAGAAT-3'(SEQ ID NO.6)；
(II) extraction of genomic DNA
Cutting part of tail fin of Lateolabrax japonicus about 0.5 × 0.5cm, and storing in anhydrous alcohol at room temperature. Extracting the DNA of the fin-shaped genomic DNA (marine animal tissue genomic DNA extraction kit (Tiangen)), detecting the quality of the genomic DNA by adopting 1.0 percent agarose gel electrophoresis, detecting the concentration by utilizing an ultraviolet spectrophotometer (Eppendorf, AG2231 type), and storing at-20 ℃ for later use.
(III) multiplex PCR amplification
1. The primers were dissolved in 1 XTE to 10pmol, and primers in one set were added to the primer, mixed well and centrifuged. The primary PCR system was as follows:
the PCR mixture prepared above was dispensed into a 96-well PCR plate, centrifuged, and 2. mu.l of DNA sample was added to each well, and centrifuged.
The primary PCR amplification reaction program is as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 15s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 30s, and 35 cycles; extension at 72 ℃ for 3 min.
Purification of PCR products
3 μ l of PCR amplified product was purified by ExoI, which mainly functions to remove the remaining primers in the reaction product, and FastAP, which mainly functions to remove the remaining dNTPs in the reaction product. The system is as follows:
the reaction conditions are as follows: the extension reaction is carried out after purification at 37 ℃ for 15min and at 80 ℃ for 15min, and extension primers are mixed in advance.
3. Extension reaction
The system is as follows:
the reaction conditions are as follows:
4. mu.l of extension primer was added to 10. mu.l of Hidi sample, denatured at 95 ℃ for 3min, immediately subjected to ice water bath, and loaded onto sequencer.
Detecting the size and the peak color of the extension product on a sequencer, and determining that the SNP locus at the 202 nd base of the genome sequence SEQ ID NO:1 of the NCCRP-1 gene of the sample to be detected is GG or TT, if GG, Florida subspecies, and if TT, North subspecies.
And/or whether the 76 th base insertion mutation site of the genome sequence SEQ ID NO:1 of the NCCRP-1 gene of the sample to be detected is AA or- - (wherein "-" represents base deletion), if yes, the NCCRP-1 gene is Florida subspecies, and if yes, the NCCRP-1 gene is northern subspecies.
And/or whether the 132- -.
The SNaPshot SNP typing result of the invention is consistent with the sequencing result of a PCR product, and the SNP locus of the invention can provide reliable basis for the identification of northern subspecies of micropterus salmoides and Florida subspecies. When the number of samples is small, the northern subspecies of the largemouth bass and the Florida subspecies can be directly identified by PCR product sequencing, and the method is quick and accurate; when the number of samples is large, northern subspecies of the largemouth bass and the Florida subspecies can be identified by the SNaPshot method, the cost is low, and the method is suitable for popularization and application.
In conclusion, the inventor researches and discovers that a SNP site is found at the 202 nd base of the genomic sequence SEQ ID NO. 1 of the NCCRP-1 gene of the largemouth bass, and one and two insertion deletion mutation sites are respectively found at the 76 nd base and the 132 nd 133 nd base. And the genotype at the 76 th and 132 th along with 133 th sites of the genome sequence of the NCCRP-1 gene of the largemouth bass is found to be completely linked with the SNP at the 202 nd site, namely, when the SNP site at the 202 nd site GG is adopted, the genotype at the 76 th site and the 132 th along with 133 th site are respectively- - - - - - - - - - - -, AAAA ("- -" represents base deletion); when the SNP site TT at position 202 is found, the genotypes at positions 76 and 132 and 133 are AA and minus (- "- - - - -" indicates that the base is deleted), respectively.
In 5 groups detected by the invention, the 202 nd SNP locus genotype is taken as an example, the northern subspecies are all TT genotypes, and the Florida subspecies are all GG genotypes, which are consistent with the previous research results, and the SNP locus can rapidly and accurately identify the northern subspecies of the largemouth bass and the Florida subspecies.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> Zhujiang aquatic research institute of aquatic science research institute of China, Guangdong Liang aquatic specie industry Co., Ltd
<120> SNP marker related to identification of northern subspecies and Florida subspecies of micropterus salmoides and application thereof
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tcaccccagg tccatagcca gagaacacat gtgacacctg caggccaaga gagagacata 420
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Claims (9)
1. The genome sequence of the largemouth bass NCCRP-1 is shown in SEQ ID NO:1 and is characterized in that,
the 76 th mutation is A or base deletion,
the 132-th 133-bit mutation is AA or base deletion,
k at position 202 is a base G or T.
2. The identification of the related SNP site or insertion deletion mutation site of the largemouth bass species is characterized in that the SNP site is positioned at the 202 nd position shown by the NCCRP-1 genome sequence SEQ ID NO:1, and the insertion deletion mutation site is positioned at the 76 th position and the 132 th position 133 th position shown by the NCCRP-1 genome sequence SEQ ID NO: 1.
3. The use of the SNP site or the insertion deletion mutation site according to claim 2 for identifying a largemouth bass species.
4. The use of claim 3, wherein said micropterus salmoides are micropterus salmoides northern subspecies and Florida subspecies.
5. A method for identifying northern subspecies and Florida subspecies of micropterus salmoides is characterized by comprising the following steps:
detecting that the SNP locus at the 76 th base of the genome sequence SEQ ID NO of the largemouth bass NCCRP-1 is AA or-genotype, wherein "-" represents base deletion, if the "-" is, the fingerling is a Florida subspecies, and if the "-" is AA, the fingerling is a northern subspecies;
or/and detecting that the SNP locus at the 132-133 th base of the genome sequence SEQ ID NO:1 of the largemouth bass NCCRP-1 is AAAA or-genotype, wherein, the-represents base deletion, if the SNP locus is AAAA, the SNP locus is Florida subspecies; if it is "- - - -", it is northern subspecies;
or/and detecting that the SNP locus at the 202 nd base of the genomic sequence SEQ ID NO:1 of the largemouth bass NCCRP-1 is GG or TT genotype, if GG, Florida subspecies, and if TT, North subspecies.
6. The method of claim 5, comprising the steps of:
1) extracting DNA of micropterus salmoides;
2) the extracted DNA is taken as a template, PCR is carried out to detect whether the genotypes of the 76 th site, the 132 th site and the 202 th site of the genomic sequence SEQ ID NO. 1 of the largemouth bass NCCRP-1 are AA or- -, AAAA or- - -, GG or TT genotypes respectively, wherein "-" represents the base deletion.
7. The method according to claim 6, wherein the PCR detection in step 2) is specifically performed by:
carrying out PCR amplification on the DNA of the largemouth bass by using primers P1 and P2 to obtain a PCR product,
P1:5'-AAGCACAAAGTGTAGGTATTTTCCA-3'；
P2:5'-GGCTGCGGATAAAAGCACTG-3'；
sequencing and analyzing the obtained PCR product to determine whether the 202 nd SNP site genotype of the genome sequence of the largemouth bass NCCRP-1 gene is GG or TT genotype and whether the 76 nd and 132 nd 133 nd insertional deletion mutation sites are AA or- - -, AAAA or- - -genotype respectively.
8. The method of claim 7, wherein the PCR amplification reaction system is:
9. the method of claim 8, wherein the PCR amplification reaction procedure is: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, and 35 cycles; extension at 72 ℃ for 10 min.
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| CN113637765A (en) * | 2021-07-16 | 2021-11-12 | 中国水产科学研究院珠江水产研究所 | Molecular marker for identifying genetic sex of micropterus salmoides and application |
| CN113637765B (en) * | 2021-07-16 | 2023-07-21 | 中国水产科学研究院珠江水产研究所 | Molecular marker for identifying genetic sex of micropterus salmoides and application thereof |
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